310 Data Collection Software • Controls 310 run conditions • Translates light on CCD camera into electropherogram (raw data) • Sample sheets and injection lists are created Macintosh 1.0.2 1.2.2 2.1 (5-dye) Windows NT Just being released ABI manual is P/N 904958B
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310 Data Collection Software - Strbase · 310 Data Collection Software • Controls 310 run conditions • Translates light on CCD camera into electropherogram (raw data) • Sample
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310 Data Collection Software
• Controls 310 run conditions• Translates light on CCD camera into
electropherogram (raw data)• Sample sheets and injection lists are created
Macintosh
1.0.2
1.2.2
2.1 (5-dye)
Windows NT
Just being released
Just being released
ABI manual is P/N 904958B
Injection List in Data Collection Software
• Lists samples to be analyzed (repeats can be easily performed)
• Sets virtual filter on CCD camera• Sets electrophoresis time and voltage• Sets injection time and voltage• Sets run temperature• If desired, sample analysis can be set up for
automatic matrix color separation and sizing with internal standards using defined analysis parameters
Steps Performed in Standard Module
• Capillary fill – polymer solution is forced into the capillary by applying a force to the syringe
• Pre-electrophoresis – the separation voltage is raised to 10,000 volts and run for 5 minutes;
• Water wash of capillary – capillary is dipped several times in deionizedwater to remove buffer salts that would interfere with the injection process
• Sample injection – the autosampler moves to position A1 (or the next sample in the sample set) and is moved up onto the capillary to perform the injection; a voltage is applied to the sample and a few nanoliters of sample are pulled onto the end of the capillary; the default injection is 15 kV (kilovolts) for 5 seconds
• Water wash of capillary – capillary is dipped several times in waste water to remove any contaminating solution adhering to the outside of the capillary
• Water dip – capillary is dipped in clean water (position 2) several times• Electrophoresis – autosampler moves to inlet buffer vial (position 1) and
separation voltage is applied across the capillary; the injected DNA molecules begin separating through the POP-4 polymer solution
• Detection – data collection begins; raw data is collected with no spectraldeconvolution of the different dye colors; the matrix is applied duringGenescan analysis
(prior to separation of fluorescent dye colors)
Raw Data from the ABI Prism 310
GeneScan® Software
• Calls peaks (based on threshold values)• Separates colors with matrix file• Sizes peaks with internal size standard
Macintosh
2.1
3.1
3.1.2 (5-dye)
Windows NT
3.7 (5-dye)
ABI manual is P/N 4303189
Screens in GeneScan® Program• Processed data• Sizing data• Electrophoresis history• Sample Information• Raw data• Analysis log file
Each screen can be used to aid in evaluation of samples and trouble shooting problem samples during data analysis
Each screen can be used to aid in evaluation of samples and trouble shooting problem samples during data analysis
Matrix Standards (Raw Data)
6FAM
TET
HEX
ROX
Save 4 x 4 Matrix Created
DNA fragment peaks in sample
DNA Size
Data Point
147.32 bp147.32 bp
165.05 bp165.05 bp
100
139150
160
200
250
DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard
Process of Sizing DNA Fragments Using an Internal Standard
Sizing Algorithm• Local Southern is commonly used but may not be
the best in all situations• Local Southern involves using 2 peak above and 2
peaks below an unknown peak from the internal size standard to make a calculated DNA size
Thoughts on Size Standards• Be consistent in use if you want to be able to
compare data over time• All size standards I have tested work• Allele sizes are different with different internal
sizing standards• GS500 has a large “hole” in its sizing ability when
using the local Southern algorithm for medium-sized STR alleles because of the 250 bp peak that cannot be used; also must be run out to 450 bp to be able to type large FGA alleles with ABI kits
Genotyper Software
• Converts GeneScan sized peaks into genotype calls using macros
• Genotyping performed by comparison of allele sizes in allelic ladder to sample alleles
Macintosh
2.0
2.5
2.5.2 (5-dye)
Windows NT
3.7 (5-dye)
ABI manual is P/N 904648
Profiler Plus Allelic Ladders
D3S1358 FGAVWA
AMEL D8S1179 D21S11 D18S51
D5S818 D13S317 D7S820
COfiler Allelic Ladders
D3S1358
AMEL
D7S820
D16S539
TH01TPOX CSF1PO
SGM Plus Allelic Ladders
D3S1358 D16S539VWA
AMEL D8S1179 D21S11 D18S51
D19S433 TH01 FGA
D2S1338
Identifiler Allelic Ladders
D3S1358 D16S539
VWA
AMEL
D8S1179D21S11
D18S51
D19S433
D5S818FGA
D2S1338
TPOX
TH01 D13S317
CSF1POD7S820
PowerPlex® 16 Ladders
D3S1358
D16S539
VWA D8S1179
D21S11 D18S51
FGA
Penta D
TPOX
D13S317CSF1PO
D7S820
AMEL
Penta ETH01
D5S818
Data Collection
Peak Identification
Data Review by Analyst/Examiner
Color Separation
Peak Sizing
Comparison to Allelic Ladder
Confirmation of Results by Second Analyst/Examiner
Genotype Assignment to Alleles
GeneScansoftware
Genotypersoftware
Internal sizing standard
(e.g., GS500-ROX)
Matrix file
Allelic ladder sample
Steps in STR Genotyping ProcessData Collection
software
Expert Systems under Development
(e.g., True Allele)
Three Possible Outcomes• Match – Peaks between the compared STR profiles have
the same genotypes and no unexplainable differences exist between the samples. Statistical evaluation of the significance of the match is usually reported with the match report.
• Exclusion – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources.
• Inconclusive – The data does not support a conclusion as to whether the profiles match. This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that i ffi i t i f ti i t t t l i
ABI 3100 16-capillary array ABI 310
single capillary
Increasing Sample Throughput with Parallel Processing
Subtle differences in matrix formation and sizing algorithms –
NOT directly equivalent to 310
Subtle differences in matrix formation and sizing algorithms –
*Data used with permission from American Association of Blood Banks (AABB) 1999 Annual Report.
Summary of STR Mutations
• Mutations happen and need to be considered• Usually 1 in ~1000 meioses• Paternal normally higher than maternal• VWA, FGA, and D18S51 have highest levels• TH01, TPOX, and D16S539 have lowest
levels
General Information•Intro to STRs (downloadable PowerPoint)
•STR Fact Sheets
•Sequence Information
•Multiplex STR Kits
•Variant Allele Reports
Forensic Interest Data•FBI CODIS Core Loci
•DAB Standards
•NIST SRM 2391
•Published PCR Primers
•Y-Chromosome STRs
•Population Data
•Validation Studies
Supplemental Info•Reference List
•Technology Review
•Addresses for Scientists
•Links to Other Web Sites
http://www.cstl.nist.gov/biotech/strbase
1479
Short Tandem Repeat DNA Internet Database
STRBase
D19
AMEL
D3
D8 VWATH01
D21FGA D16 D18
D2
D19AMEL D3
D8 VWATH01
D21
FGA
D16
D18 D2
15 years old (room temp storage)
6 years old (-20 oC storage)
Results with SGM Plus STR kit (Applied Biosystems)
“Decay curve” of degraded DNA
Degraded DNA Results
D5S818 D13S317
D7S820
D8S1179 D21S11 D18S51
Amel
VWA FGAD3S1358 blue panel
green panel
yellow panelRel
ativ
e Fl
uore
scen
ce U
nits
Sample Mixture ExampleProfiler Plus data
Higher than expected stutter
Higher than expected stutter
“Stutter” on wrong side of allele
“Stutter” on wrong side of alleleImbalance in X
and Y peak ratiosImbalance in X
and Y peak ratios
4 peaks at a single locus4 peaks at a single locus